Epirubicin (EPI) is an anti-cancerous chemotherapeutic drug that is an effective epimer of doxorubicin with less cardiotoxicity. Although EPI has fewer side effects than its analog, doxorubicin, this study aims to develop EPI nanoparticles as an improved formula of the conventional treatment of EPI in its free form. Methods: In this study, EPI-loaded polymeric nanoparticles (EPI-NPs) were prepared by the double emulsion method using a biocompatible poly (lactide) poly (ethylene glycol) poly(lactide) (PLA–PEG–PLA) polymer. The physicochemical properties of the EPI-NPs were determined by dynamic light scattering (DLS), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), entrapment efficiency and stability studies. The effect of EPI-NPs on cancer cells was determined by high throughput imaging and flow cytometry. Results: The synthesis process resulted in monodisperse EPI-NPs with a size of 166.93 ± 1.40 nm and an elevated encapsulation efficiency (EE) of 88.3%. In addition, TEM images revealed the spherical uniformness of EPI-NPs with no aggregation, while the cellular studies presented the effect of EPI-NPs on MCF-7 cells’ viability; after 96 h of treatment, the MCF-7 cells presented considerable apoptotic activity. The stability study showed that the EPI-NPs remained stable at room temperature at physiological pH for over 30 days. Conclusion: EPI-NPs were successfully encapsulated within a highly stable biocompatible polymer with minimal loss of the drug. The used polymer has low cytotoxicity and EPI-NPs induced apoptosis in estrogen-positive cell line, making them a promising, safe treatment for cancer with less adverse side effects. Epirubicin (EPI) is a chemotherapy anthracycline drug that is less cardiotoxic and more effective than its widely used derivative, doxorubicin ; hence, The main disadvantage of conventional cancer therapies is nonspecific binding, which leads to an inadequate drug amount being delivered to the tumor tissue while also causing adverse effects on normal cells. Due to their unique properties, nanoparticles (NPs) are being used to deliver chemotherapies to cancer cells . Their sFurthermore, the exceptional properties of nano-based drug delivery systems (DDSs) enable them to be formulated in different dosage forms requiring various stability properties ,29,30,31The synthesis of anti-cancer DDSs is attracting the attention of many scientists; EPI, specifically, has been encapsulated in marine carbohydrate as a kind of pH-triggered DDS . EpirubiFor nanoparticles’ synthesis, polylactide-block-poly (ethylene glycol)-block-polylactide , poly  (PVA) , and chloroform (99.0–99.4% (GC)) were purchased from Sigma–Aldrich Chemical Co. . The drug EPI hydrochloride (HCl) (molecular weight: 579.98 Da) , was dissolved in dimethyl sulfoxide (DMSO); ACS . Ultrasonication was performed using an ultrasonic processor .2 humidified incubator . For flow cytometry, an Annexin V-FITC apoptosis detection kit (BMS500FI-100|) was purchased from Invitrogen . For the stability study, the solutions used included potassium hydroxide (KOH) , Tris HCl , and phosphate buffer saline (PBS)  (Thermo Fisher Scientific). The solutions’ pH were measured with the Seven Compact S220 pH meter . All other solutions and chemicals were of analytical grade.MCF-7 (ATCC HTB-22) breast cancer cells were purchased from the American Type Culture Collection  and cultured in Dulbecco’s Modified Eagle Medium (DMEM) 1× + GlutaMax™ containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, then harvested with stable trypsin replacement enzyme (TrypLE™ Express (1×)); all were purchased from . Cells were incubated at 37 °C in a 5% CO2O) was added slowly to the solution and the sample was ultrasonicated using the same previous settings to create the double emulsion. The final formed nanosuspension was stirred for one hour at room temperature (RT) under a fume hood to facilitate the complete evaporation of chloroform. The sample was then centrifuged  in an Eppendorf tube at 14,000 rpm for one hour at RT. The precipitated NPs were next washed with 2 mL of distilled water and recentrifuged for 30 min at the same settings. After collecting the supernatant, the NPs were air-dried overnight under a fume hood. For the control, void NPs were prepared by adding free DMSO instead of the drug using the same exact method.The NPs were prepared using a double emulsion method. An amount of 40 mg of PLA–PEG–PLA polymer was dissolved in 2 mL of chloroform and 100 µM of EPI dissolved in DMSO was subsequently added. To create the first emulsion, the sample was placed in an ice bath and ultrasonicated for five minutes  at 65% amplitude. Then, 3 mL of 1.5% PVA (prepared in advance by dissolving PVA powder in dHThe average particle size and polydispersity index (PdI) were determined by dynamic light scattering (DLS) using a particle size analyzer  with an angle of detection of 90°. Both the particle size and PdI were measured five times consecutively. The average of the five instrument runs for each was calculated. The statistical analysis of particle size and polydispersity index were performed through Particle Solutions Software-Brookhaven Instruments, NY, USA.Using the aforementioned particle size analyzer , the zeta-potential of the EPI-NPs was measured by enacting the laser Doppler velocimetry mode. The statistical analysis of zeta potential was performed through Particle Solutions Software-Brookhaven Instruments, NY, USA.TEM images were collected using a JEM-1400 electron microscope  operating at an acceleration voltage of 120 kV. A drop of the sample (1 mg/mL) was placed on a 400-mesh, carbon-coated copper grid. The samples were air-dried at RT prior to recording measurements.The supernatant collected after the centrifugation processes  made from cellulose membrane, Mwt cut off = 14,000 . The sample-containing tube was immersed in 14 mL of PBS in a small dark glass bottle containing magnet. The bottle was then closed and placed on a magnetic stirrer at a speed of 4 rpm and temperature of 37 °C. To determine the amount of the released EPI, UV absorbance was measured; each time, 1 mL of the sample was taken for measurement and replaced by PBS to maintain sink conditions. The data were obtained through SoftMax Pro Software-Molecular Devices, CA, USA.At this stage, 200 µL of void NPs and 200 µL of EPI-NPs were dispersed in 800 µL of the following five different solutions for analysis: ultra-pure water (pH: 7.02), PBS (pH: 7.15), DMEM media with FBS and penicillin–streptomycin (pH: 7.10), HCl (pH: 3.26), and KOH (pH 14.05). The final concentrations of the void NP and EPI-NP dispersions were 2.2 mg/mL and 7.671 μM. The NP dispersions were stored in sealed Eppendorf tubes in the dark at RT. The stability of the NPs was tested daily in all solutions for a duration of 30 days and using the particle size analyzer , the average of the five instrument runs for each parameter  was calculated.The thermodynamic properties of EPI-NPs were studied by differential scanning calorimetry  to identify the purity degree of EPI HCl and the level of epirubicin–copolymer interaction. The DSC system was calibrated using the indium calibration standard. Then, a small amount (5–7 mg) of pure EPI, EPI-NPs, and void NPs were weighed in the DSC aluminum pans separately to be analyzed in three separate runs. The starting temperature was 30 °C and was gradually increased up to 250 °C at a rate of 10 °C per minute using nitrogen as a purging gas at a flow rate of 40 mL/min. g, 5 min, RT); this step was repeated twice. After that, 5 μL of annexin V-FITC was added to the cell suspension; subsequently, cells were incubated for 10 min at RT and then washed with binding buffer. Next, 10 μL of propidium iodide (20 μg/mL) was added to the cells’ suspension and the cell viability was determined by fluorescence-activated cell sorting (FACS), performed using the FACS Canto II flow cytometry system .An annexin V-FITC apoptosis staining was used to evaluate cell viability as per the manufacturer’s recommendations. Briefly, MCF-7 cells at passage number 12 were seeded (0.4 million) in a T25 culture flask in 3 mL of 10% FBS DMEM complete medium. After 90 min of incubation, cells were treated with EPI  of EPI-NPs. As a control, the cells were also treated with EPI in its free form at the same concentrations. In addition, two T25 flasks containing binding buffer and annexin-V were used as controls. After 90 min of treatment, supernatant and attached cells were collected by centrifugation. The collected cells were washed with PBS, then centrifuged , HOECHST33342 (5 µg/mL), and propidium iodide (2.5 µg/mL) for 20 min at 37 °C and 5% CO2. Then, cells were imaged using the ImageXpress® Micro system and analyzed with the MetaXpress® software . Nuclei were counted in each well and the average fluorescence intensity was calculated. All experiments were performed in triplicate and their outcomes were averaged; resultant values were reported as the mean ± standard deviation.MCF-7 cells were plated in 96-well plates at a density of 10,000 cells per well in an appropriate amount of DMEM media. Cells were treated with free EPI , EPI-loaded NPs , and void NPs for zero, 24, 48, 72, and 96 h. The cells were incubated at 37 °C under the condition of 5% COp-value < 0.05. Statistical operations and calculations were performed using Microsoft® Excel® 2016.Data for the stability study were expressed as the mean of five independent experiments ± standard deviation (SD). Linear regression analysis was performed to describe the relationships between a set of independent variables and the dependent variable. Ordinary least squares (OLS) regression tests were performed to compare two groups of quantitative variables: days (X) and zeta potential (Y) and days (X) and particle size (Y). The significance of the results was at the level of Here, the synthesis of EPI-NPs is described. The EPI-NPs were prepared using a previously reported method ,41. The The EPI-NPs were synthesized successfully via the previously described double emulsion method . Figure TEM was used to determine the morphology of the synthesized EPI-NPs. co-glycolic acid (PLGA) and they used several surfactants to increase the encapsulation of EPI [In this study, the synthesis of the EPI-NPs was optimized to achieve a high encapsulation efficiency, thus improving the biopharmaceutical properties, to ensure an enhanced efficacy of EPI. The encapsulation efficiency was obtained by measuring EPI UV absorbance (for both the encapsulated drug and the excess in the supernatant); the %EE was 82% from both measurements. The EE mainly depends on the polymer composition, drug solubility and functional groups. The use of PVA as a surfactant maintains the stability of the emulsion, specifically during solvent evaporation. Some studies have shown that the use of PLA–PEG copolymers and its end-group derivative nanoparticles has the advantage of increasing drug loading and entrapment efficiency. This can be obtained by adjusting the PEG/PLA ratio to increase the efficiency of hydrophobic drugs ,48. Furtn of EPI .The stability and aggregation behavior of the EPI-NPs and void NPs in different solutions were evaluated. Measurements were taken at different time points over a duration of 31 days. This study evaluates the influence of pH and different solutions on the stability of the EPI-NPs. The stability of the EPI-NPs was evaluated in different pH solutions. The different pH solutions used in this study covered the different environments the NPs might encounter if used in vivo. The pH ranged from acidic, physiological pH and basic pH values; the information reported from this study will determine how to best store EPI-NPs to maintain maximum efficacy ,44.The surface charges of the NP suspension were assessed by measuring the ƺ . GeneralRegarding the particle size , the EPIIn regard to the polydispersity index, PdI,  EPI-loadFor the void NPs, the PdI of NPs in KOH and HCl suspensions indicated polydispersity and significantly increased during the study . For NPs in the water suspension, a slight shift toward the polydisperse range (day 0: 0.24 vs. day 31: 0.32) was observed, while, for NPs in DMEM media suspension, a shift toward the monodisperse range was observed (day 0: 0.60 vs. day 31: 0.36). In a similar way to EPI-loaded NPs, after the initial addition of void NPs to PBS, the PdI indicated high polydispersity (1.18) that decreased (0.16) over the course of the study. The PdI results confirm that stable monodispersed NPs remained monodispersed and non-aggregated even after 30 days of synthesis.The DLS results indicated that the NPs were sufficiently stable during 31 days of storage in the dark at RT . In geneThe increase in size of the EPI-NPs immersed in DMEM media can be attributed to the adsorption of FBS found in the media. Following the cross-linkage of polymeric NPs with bovine serum albumin (BSA), Palanikumar et al. analyzed the 26 most abundant serum proteins and found that PLGA NPs had higher adsorption than the BSA-PLGA NPs did . HoweverIn addition to the pH values, many factors can cause NPs aggregation, including salts and enzymes . This maThe thermodynamic properties of EPI-NPs were studied by differential scanning calorimetry  to identify the purity degree of EPI HCl and the level of epirubicin–copolymer interaction. The DSC system was calibrated using the indium calibration standard. Then, a small amount (5–7 mg) of pure EPI HCl, EPI-NPs, and void NPs were weighed in the DSC aluminum pans separately to be analyzed in three separate runs. The starting temperature was 30 °C and was gradually increased up to 250 °C at a rate of 10 °C per minute using nitrogen as a purging gas at a flow rate of 40 mL/min. The DSC thermograms are presented in The EPI-NPs’ thermogram presented in Flow cytometry has been used in this study to evaluate the effect of EPI-NPs on the MCF-7 cell line and to compare it with the effect of free-form EPI on the same cells . The MCFFlow cytometry was used in this study to evaluate the effect of EPI-NPs on the MCF-7 cell line and to compare it with the effect of free-form EPI on the same cells. The MCF-7 cells were treated with 6 nM, 12 nM, 24 nM, and 48 nM of EPI-NPs and 6 nM, 12 nM, 24 nM, and 48 nM EPI for 90 min. Different concentrations of EPI-NPs were incubated with MCF-7 cells to identify necrotic cells by staining with HOECHST33342/PI staining. The high-content imaging experiment showed that treatment with EPI-NPs causes cell death as compared with the control and void-NPs treatments. EPI-NPs were successfully synthesized and optimized. The EPI-NPs were characterized by different methods. They possess a relatively small particle size, which allows them to be internalized and delivered in cancerous tissue. With the optimized method of synthesis, the EPI-NPs had an encapsulation efficiency of around 82%, which proves the efficient synthesis method used for the preparation. The flow cytometry studies showed that the EPI-NPs have an apoptotic effect on MCF-7 breast cancer cells. In addition, the high-content imaging studies revealed a gradual decrease in cancer cells number after treatment with EPI-NPs. The stability of EPI-NPs was intensively investigated to determine their potential use as DDSs. In addition, the EPI-NPs showed a sustained drug release profile, and they were stable at different pH values and in different conditions. However, they were mostly stable as physiological pH values, which makes them prime candidates for different pharmaceutical dosage forms. Further in vivo studies are recommended as a next step to study the pharmacokinetics of the EPI-NPs in animal models.


Interstitial brachytherapy (BT) is generally used for the treatment of well-confined solid tumors. One example of this is in the treatment of prostate tumors by permanent placement of radioactive seeds within the prostate gland, where low doses of radiation are delivered for several months. However, successful implementation of this technique is hampered due to several posttreatment adverse effects or symptoms and operational and logistical complications associated with it. Recently, with the advancements in nanotechnology, radioactive nanoparticles (radio-NPs) functionalized with tumor-specific biomolecules, injected intratumorally, have been reported as an alternative to seed-based BT. Successful treatment of solid tumors using radio-NPs has been reported in several preclinical studies, on both mice and canine models. In this article, we review the recent advancements in the synthesis and use of radio-NPs as a substitute to seed-based BT. Here, we discuss the limitations of current seed-based BT and advantages of radio-NPs for BT applications. Recent progress on the types of radio-NPs, their features, synthesis methods, and delivery techniques are discussed. The last part of the review focuses on the currently used dosimetry protocols and studies on the dosimetry of nanobrachytherapy applications using radio-NPs. The current challenges and future research directions on the role of radio-NPs in BT treatments are also discussed. Cancer is one of the main causes of human death worldwide . Along wClinical trials and preclinical studies using BT have reported promising outcomes. However, the logistical and operational difficulties associated with BT seed placement have impeded its successful application. For instance, in patients with prostate tumor, the transrectal ultrasound (TRUS)-based implantation approach is used to implant radioactive seeds within the tumor , 9. The 225Ac  as an unconventional BT procedure, involving intratumoral injection of these radiolabeled AuNPs. Such nanoparticle-based systems i) conserve the characteristics of BT, i.e., precise and targeted dose delivery; ii) can be administered through injection; and iii) have the ability to provide patient-specific treatment, as radiation dose can be divided into several fractions. Additionally, these radiopharmaceuticals do not need seed removal; hence, they can be handled easily and can be extremely useful. The nanometer size of these radiopharmaceuticals allows local diffusion from the site of injection and may result in homogeneous dose distribution within the tumor volume. Lastly, these nanomaterials (particularly high Z nanoparticles) can be used as multifunctional carriers to deliver radioisotopes to provide imaging and RT capabilities. Such radioactive high-Z nanoparticles may also enhance radiation dose through self-sensitization and may require less radioactivity in comparison with conventional BT.Recently, several preclinical studies on localized delivery of radioactive nanoparticles (radio-NPs) into the tumor, similar to BT, have been reported in the literature, and this technique is termed as nanobrachytherapy –13. In nIn this article, we review the recent advancements in the synthesis and use of radio-NPs as nanobrachytherapeutic agents. The subsequent section presents a review and discussion on different techniques involved in radiosynthesis of nanoparticles. The particles emitted by radionuclides, present in the obtained radionuclide–nanoparticle complex, must deposit their energy locally and spare the surrounding normal tissues. Hence, in the succeeding section, the essential characteristics of radionuclide–nanoparticle complexes, which are vital to qualifying them as nanobrachytherapeutic agents, are discussed. After intratumoral injection, these radio-NPs diffuse 1–2 mm within the extracellular medium, from the site of injection , and arein vivo pharmacokinetics —allows for specific targeting of receptors overexpressed on tumor cells or within the tumor microenvironmentRadionuclide selection (imaging and/or therapeutic)—emission mode, decay half-life, and chemical properties, availability, and radiolabeling reactionRadiolabeling strategy to achieve the maximal radiochemical purity and yield, which reflects specific activity of nanoradiopharmaceuticalsAmong the different types of nanoparticles, AuNPs and iron oxide nanoparticles (IONPs) have gained more prominence due to their superior biocompatibility, low toxicity, ease in surface versatile functionalization and radiolabeling with a plethora of imaging, and therapeutic radionuclides towards the development of nanoradiopharmaceuticals for imaging and therapy of cancer. Translational medicine that makes use of nanoradio-pharmaceutical agents demonstrates excellent pharmacokinetics in terms of radiochemical production, purity and stability (nanoradioformulation integrity), biodistribution, dosimetry, low off-target localization, and favorable renal clearance profiles, which represent a versatile theranostic tool in cancer management, ranging from nuclear medicine imaging and image-guided surgery to alpha/beta-particle targeted therapy, and most recently targeted nanobrachytherapy –21. The via coordination chemistry approaches labeling strategies for nanoparticles are available for the development of multimodal nanoradiotherapeutics  as shownproaches , 20, 24:Bifunctional moieties that provide capping/stabilizing capabilities with subsequent binding affinity to the radioisotopesDirect surface conjugation of amino/thiolated molecules followed by ligand exchangeChemical modification of molecules already attached on the surface of the nanoparticlesvia exogenous coordination chemistry moieties [bifunctional chelators (BFCs) and prosthetic groups] through chemical linkers to aid complexation . Among the radionuclides, 177Lu (t1/2 = 6.734 days) with both β emissions and γ rays is of interest for theranostics. 177Lu entrapping AuNPs inside the dendritic cavity of a generation 4 (G4) polyamidoamine (PAMAM) dendrimer, which had been pre-conjugated with p-SCN-benzyl-DOTA as well as folate/bombesin for cancer targeting (64Cu (64Cu-DOTA-mAb) for positron emission tomography (PET) imaging utilized to estimate tumor density, perfusion, and distribution in mice bearing MDA-MB231 anti-programmed death-ligand 1 (PD-L1-positive) xenograft and HER2-targeted antibodies for patients with metastatic HER2-positive breast cancer (BC)  with radiochemical purity >98%. Most recently, AGuIX® represents gadolinium (67Gd)-DOTAGA cyclic chelates covalently grafted to polysiloxane matrix to produce AGuIX nanoparticles , is the most commonly utilized BFC owing to its high affinity to most metal radionuclides (argeting . Cancer cer (BC) , 28. Polhelation . Hajiramhelation  developearticles , 31.3O3 chelator, 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), and its derivative are commonly used for gallium and copper radiopharmaceuticals (67Ga/68Ga and 64Cu) for radiolabeling nanoparticles. The general approach for conjugating nanoparticles with NOTA moiety for 67/68Ga and 64Cu labeling is through thiol-functionalized NOTA (NOTA-SH) for radiolabeling and conjugation, additionally linkers/spacers such as polyethylene glycol (PEG) and PEI to optimize in vivo pharmacokinetics. NOTA-SH can be achieved by reacting p-SCN-Bn-NOTA with 2-aminoethanethiol hydrochloride in the presence of triethanolamine.A hexadentate N99mTc, 111ln, and 67/68Ga). However, the DTPA complex exhibits low in vivo kinetic stability characterized by fast dissociation kinetics and radiometal complexation, and the functionalization of nanoparticles with polymers such as PAMAM and PEI improved stability.A polydentate acyclic chelator, diethylenetria-minepentaacetic acid (DTPA), is commonly used in the construction of MRI and nuclear imaging agents  (18F]-Fluoro-2-deoxy-d-glucose (18F-FDG) is used for the assessment of glycolysis as a non-invasive PET imaging agent. In an archetypical example, radiolabeling nanoparticles with 18F has been reported by first conjugating cysteamine to mannose triflate (Man-CA) and then 18F labeling resulting to a cysteamine-linked radiotracer (18F-FDG-CA). The 18F-FDG-CA is mixed with gold chloride (HAuCl4) to obtain AuNPs (18F-FDG-CA-AuNPs) (Indirect radiolabeling A-AuNPs) .Ionophore-based radiolabeling is divided into subclasses: i) ionophore-chelate binding and ii) remote loading radiolabeling. Both ionophore-chelate binding and remote loading radiolabeling use lipophilic radiotracers with passive lipid membrane permeability properties . RadiolaIndirect radiolabeling using chelator-based  has gained prevalence in nuclear medicine. The negative impact on the biological activity of the overall radiolabeled nanoparticles is attributed to changes in the size, surface charge, and hydrophilicity of the nanoparticles. This problem can be overcome through direct and chelator-free radiolabeling strategies while maintaining the nanoparticle’s native pharmacokinetic characteristics.via electrostatic attraction or van der Waals interactions (99mTc and 177Lu) to produce 99mTc@AuNCs and 177Lu@AuNCs, respectively, as a novel approach for tumor radio-immunotherapy.Chemisorption is facilitated by mixing the radionuclides with nanoparticles that exhibit high binding affinity towards the radionuclides for direct chemical bond formation between the surface of the nanoparticles and the radionuclide. This is achieved through the oppositely charged moieties on the surface of the nanoparticles and the radionuclide, thus allowing for chemical adsorption. Likewise, physisorption occurs when charged radionuclide ions interact with the molecular surface of the nanoparticles ractions . Pei et ractions  designed64Cu or 111ln), and homo-radionuclides, where a radioisotope of the metal element to form the nanoparticle core is used   by intrinsically radiolabeling during synthesis of AuNPs through the use of H199AuCl4 precursor. Fach et al.  with ≈20 nm, and then ethylenediaminetetraacetic acid (EDTA) was used to scavenge free Pd2+ to avoid unspecific labeling of the nanoparticle surface resulting in radiolabeling efficiencies of 79% to >99%.Radiochemical labeling involves incorporation of the radionuclide as a surrogate during the synthesis of the nanoparticles resulting in intrinsically radioactive nanoparticles often carried out in automated closed lead-shielded unit due to the increased radiation exposure , 32. Thi98AuNPS) , 35, 36.98AuNPS)  producedy et al.  producedh et al.  doped propionate for 124I or 125I radiolabeling, followed by reacting the nanoparticles with HAuCl4 to form a Au shell to shield radionuclide dissociation, thus resulting in 124/125I-Au@AuNPs this approach was further used to produce 124I-labeled tannic acid gold core-shell nanoparticles (124I-TA-Au@AuNPs) exhibiting 98% radiochemical yield. Laan et al. (111ln-labeling polystyrene-b-poly (ethylene oxide) diblock copolymer micelles without the necessity of any chemical modification.Encapsulation is through entrapping the radionuclide inside the native cavity within the nanoparticles or within core-shell/layered structured nanoparticles. Lee et al.  demonstrn et al.  reported59Fe-labeled IONPs by oleic acid-functionalized IONPs in chloroform, and then the IONPs were incubated with 59FeCl3 which led to approximately 0.01%–0.5% 59Fe exchange with Fe3+ (homogenous) in the IONPs. The low isotope exchange of 59Fe/Fe is attributed to Fe surface availability of the IONPs. Heterogeneous radioisotopic exchange was demonstrated by Tang et al.  with >98% radiochemical purity. Zhang et al.  during decay of the latter . Howeverα, β, and Auger particle-emitting radionuclides, as reported in The radionuclides to be used for internal RT must deliver high doses of radiation locally and spare the surrounding normal tissues , 13. Henα, β, and Auger electrons or photons; c) energy of the emitted particles; d) daughter product(s) and their stability; e) radionuclide purity and length of purification step; f) penetration depth of the emitted particles in the biological tissues; g) LET of the emitted particle; and h) size of the tumor to be treated  toxicity caused by the complex  physical and ii) biochemical characteristics. The physical characteristics to be considered are a) physical half-life; b) emitted particle type— treated , 13. Add complex –55.in vivo pharmacokinetics of the radionuclide–nanoparticle complex (Tp) of the radionuclide and half-clearance time, also known as biological half-life (bT), of the radionuclide–nanoparticle complex using the relation 1/T = 1/pT + 1/bT , β emitters are regarded as the most suitable for the treatment of large or bulky tumors  in comparison with the α emitters , 59. Fory tumors . β-Emittc agents , 49, 50.tization . Photoel/tissues , 54, 55.103Pd, 111In, and 125I have been used as nanobrachytherapeutic agents in preclinical studies involving tumor-bearing xenograft models  photons and have been used for low-dose-rate BT applications since the 1970s. The emitted photons deposit up to 98% of their energy within ≈5–8 cm of soft tissue and can be used to treat large and bulky tumors. 111In also emits photons with energy greater than 200 keV and is not suitable for internal RT purposes or radiation dose enhancement through radiosensitization  with short range in the biological tissues (a few nanometers) , 55. 103t models , 45, 52.tization . In casetization .Hence, the choice of the radionuclide also depends on the size of the tumor to be treated. It is because bulky tumors, micrometastases, and a small cluster of tumor cells require particles of specific energy for effective ablation of cancer cells. Further, the mode of radiosynthesis of nanoparticles and the length of the purification step (of radionuclides) must be selected according to the half-life of the radionuclide , 55. In in vivo before it is excreted or metabolized  and Calu-3  cells. According to their study, positively charged nanoparticles cause higher levels of cytotoxicity as compared with negatively charged ones, and it might be due to the oxidative stress, mitochondrial damage, and cellular overall toxicity observed for those kinds of particles. Negatively charged particles are known to be less cytotoxic to epithelial cells, and this might be explained by the fact that those cells present a net negative charge in their extracellular portion of plasma membrane, enabling a better interaction with positively charged nanoparticles. The results obtained for other cell types are sometimes conflicting; therefore, more studies might be necessary in order to understand the mechanisms for each tissue .via a dynamin-dependent mechanism, but the inhibition of clathrin-mediated transport likely caused an increase in the transport of negatively charged particles, though with no effect on their cell uptake. Regarding positively charged particles, clathrin inhibition reduced by 46% their cell uptake and by 38% their transcellular transport, whereas micropinocytosis inhibition reduced the internalization of the same particles by 42%, and the transcellular transport by 38%, similarly to micropinocytosis inhibition by methyl-β-cyclodextrin  and the intracellular transport (48%) of negatively charged nanoparticles, leading to the assumption that caveolae-dependent endocytosis plays an important role for those nanoparticles .The protein corona is another key factor to be considered when developing any nanomaterial with biomedical applications. The protein corona is formed whenever a nanomaterial is introduced into a complex protein aqueous system and consists in the rapid adsorption of the most abundant proteins onto the surface of the nanomaterial, followed by the exchange of at least part of these proteins for others with higher affinity for the nanomaterial. The result is a nanoparticle with a completely different surface coating than that predicted in the design phase, sometimes with tendency for aggregation or with higher stability, with different internalization rates (enhanced or impaired), and different pharmacokinetics , 69.The composition of the protein corona is not universal, as it depends on the nanomaterial and on the previous coating. It was demonstrated, for instance, that citrate-coated IONPs were not stable while in contact with fetal calf serum proteins but were efficiently internalized by lymphoblastoid cells, while poly(acrylic acid)-coated IONPs were quite stable, although were poorly taken up, which can be a barrier to be faced by the nanomaterials inside the blood .Another barrier that the nanoparticles must overcome is the reticuloendothelial system (RES), responsible for a rapid clearance of the nanomaterials once they enter in the bloodstream, decreasing their pharmacological action. Strategies to avoid the clearance by RES include surface modification with molecules that prevent opsonization and increase the half-life in blood, such as PEG. However, as described previously, many variables must be added, such as surface charge. Harush-Frenkel and collaborators verified once more the preferential internalization of positively charged nanoparticles (twice the endocytosis of their negatively charged counterparts) in HeLa cells, and after 45 min, the cells tend to decrease the uptake rate, characterizing a saturation phase .Another factor that contributes to the decreased circulating time of nanoparticles in blood is the mononuclear phagocytic system, in which the macrophages quickly scavenge nanoparticles that are agglomerated or covered with the protein corona, preventing their arrival at the target site. Zhang and coworkers used the advantage of folic acid as a functionalizing agent in PEGylated superparamagnetic magnetite nanoparticles (circa 10 nm in diameter) in the internalization efficiency by mouse macrophages (RAW 264.7 cells) and human breast tumor (BT-20 cells). PEG was responsible in partially inhibiting the formation of the protein corona in order to decrease the recognition of nanoparticles by macrophages, whereas folic acid was added to the surface of nanoparticles to specifically target cancer cells overexpressing folate receptors in order to increase their uptake .The alpha-, beta-, and Auger-emitting radionuclides have been investigated for nanobrachytherapy applications. A few preclinical studies on nanobrachytherapy applications using alpha, beta, and Auger emitters have been published in literature. The most recent ones are briefly discussed in this section, and a brief summary of these studies is also presented in 211At on the surface of AuNPs. The in vitro cytotoxicity of the obtained 211At-AuNP-S-PEG-SP (5-11) radiobioconjugate was evaluated in human serum and cerebrospinal fluid. No study on therapeutic efficacy and in vivo biodistribution of radiobioconjugate has been reported. However, the authors recommended the intratumoral injection of 211At-AuNP-S-PEG-SP (5-11) radiopharmaceutical, instead of intravenous injection due to its large size.Dziawer et al.  synthesi211At chemically adsorbed on its surface for nanobrachytherapy purposes using alpha emitters [56]. The nanoparticles were activated with PEG and trastuzumab (antibody) to actively target HER-2 proteins overexpressed on the surface of ovarian cancer-derived SKOV-3 cells. In the in vitro study, the authors demonstrated that AuNP-S-PEG-trastuzumab bioconjugate was effectively internalized by SKOV-3 cells. Furthermore, an in vitro cell viability test demonstrated that 211At-AuNP-trastuzumab radiobioconjugate effectively reduced the metabolic activity of ovarian cancer cells with a median lethal dose of 0.5 MBq/mL. In this case as well, no biodistribution or therapeutic evaluation was reported.Recently, the same group synthesized 5-nm-sized AuNPs, with 225Ac via DOTA-derivative (TADOTAGA) chelator. The chelator TADOTAGA formed a strong bond with the AuNPs resulting in the formation of a highly stable colloid in aqueous medium, and the chelating characteristics of DOTA-derived macrocyclic compound were exploited to radiolabel the Au@TADOTAGA nanocarriers. The [225Ac]225Ac-Au@TADOTAGA nanoparticles (5–9 nm) were synthesized with radiochemical yield of 86% and radiochemical purity greater than 93%. The aim of the study was to evaluate [225Ac]225Ac-Au@TADOTAGA nanoparticles as a nanobrachytherapy agent. The radiolabeled nanoparticles were evaluated in terms of i) its stability and in vitro cytotoxicity in U-87 MG (human glioblastoma–astrocytoma) cancer cells and ii) in vivo biodistribution by intravenous (i.v.) and intratumoral injection of [225Ac]225Ac-Au@TADOTAGA nanoparticles to the mice bearing U-87 tumor. Additionally, the tumor regression studies were performed over a period of 22 days to evaluate the therapeutic efficacy of intratumorally injected 225Ac radiolabeled nanoparticles. For in vivo biodistribution studies, the mice (tumor volume = 200–400 mm3) were divided into two groups, with three to five mice in each group. The [225Ac]225Ac-Au@TADOTAGA  nanoparticles were injected intravenously to the first group and intratumorally to the second group. The mice were euthanized at 2, 4, 24, 72, 120, and 288 h after injection; all the major tissues and organs were removed and weighted; and radioactivity was counted in terms of % injected dose per gram (%ID/g). For the first group (i.v. injection) at 2 h post injection (p.i.), the uptake of radiopharmaceuticals in the kidney ≈28%ID/g decreased to ≈9%ID/g at 120 h p.i., which showed the renal clearance of AuNPs, whereas the uptake in the liver and spleen increased from 9.5%ID/g and 7.2%ID/g at 2 h p.i. to 21.5%ID/g and 13.3%ID/g at 120 h p.i. The maximum uptake in tumors (4%ID/g) occurred at 2 h p.i. and decreased to 1%ID/g at 120 h p.i. On the other hand, for the second group , the reported tumor uptake was 60.67%ID at 2 h p.i. and decreased to 5.2%ID/g at 228 h p.i. For therapeutic efficacy evaluation, mice with ≈300 mm3 of U-87 MG tumor xenograft were again divided into two groups. The first group (control) was injected intratumorally with 100 μL of saline, and the second group (therapy group) was injected with 100 μL/5 kBq of [225Ac]225Ac-Au@TADOTAGA on days 1, 3, and 5; and the tumor volume was tracked over 22 days. The tumor volume of therapy group was reported ≈2.4 times lower after 8 days of radioactive injection and ≈4 times lower after 22 days of injection, in comparison with the control group.Salvanou et al.  synthesi103Pd for nanobrachytherapy applications. A monodispersed layer of 103Pd was coated on gold spherical shells, hence synthesizing 103Pd@Au nanoseeds with a diameter of approximately 120 nm. These nanoseeds were injected intratumorally to mice bearing prostate cancer tumors to evaluate their in vivo therapeutic efficacy and biodistribution. The mice were randomized into three groups (n = 6), treated with phosphate-buffered saline (PBS) solution, non-radioactive (cold) Pd@Au nanoparticles in PBS suspension, and radioactive 103Pd@Au nanoparticles in PBS suspension. In order to achieve uniform distribution of radiation dose in the whole tumor mass (181.7 ± 62.1 mm3), the intratumoral injection was injected at six to nine locations, and radioactivity of 1.5 mCi per tumor was injected. The total injected volume of PBS, cold Pd@AuNPs, and 103Pd@AuNPs was kept below 40 μL. The evaluation of retention of nanoseeds within the tumor volume and their migration to other organs was performed ex vivo and with single-photon-emission CT (SPECT)/CT. Upon SPECT/CT imaging, it was reported that after day 1 of radioactive injection, 101.50 ± 23.72%ID/g was retained within the tumor volume, and a negligible amount of radioactivity (≈0.1%ID/g) was observed in the liver and spleen. Furthermore, after 5 weeks of radioactive injection, 274.5 ± 77.6%ID/g was detected in the tumor volume, as the tumor volume decreased over the course of the treatment. This indicated the expected radiotherapeutic effect of the 103Pd@Au nanoseeds. Furthermore, the ex vivo biodistribution investigation (5 weeks p.i.) results showed that ≈95% of nanoseeds were retained within the tumor, ≈3% migrated to the liver, and approximately 0.5% were found in the spleen. In terms of therapeutic efficacy, after 5 weeks of radioactive injection, the decrease in the tumor volume by about 75% for the 103Pd@Au treated group was reported, whereas the increase in the tumor volume for groups treated with PBS and cold nanoparticles was reported.Moeendarbari et al.  reported111ln of 30-nm diameter. The radionuclides (111ln) were attached to the AuNPs using DTPA. The nanoparticles were also functionalized with PEG chains linked to antibody trastuzumab. Consequently, trastuzumab-AuNP-111ln radiopharmaceutical was obtained. Trastuzumab was used to actively target HER2-positive BC cells. The authors evaluated the in vitro cytotoxicity of synthesized radiolabeled nanoparticles on HER2-positive BC cells. Additionally, in vivo therapeutic effectiveness of trastuzumab-AuNP-111ln was also assessed by intratumorally injecting 10 MBq (≈270 µCi) of radiopharmaceutical into subcutaneous HER2-overexpressing BC xenografts. Tumor growth in the BC murine model was monitored for more than 70 days post radioactive injection. Inhibition in growth of tumor was reported for the treated group, whereas in the case of the untreated group, the tumors grew up to eightfold of the initial size. Tissue toxicity was not observed. No information regarding the migration of radiolabeled AuNPs to the liver and spleen was provided, as the authors did not perform biodistribution evaluation.Cai et al.  synthesi103Pd : Pd) coated with gold (Au)-103Pd : Pd@Au and 103Pd : Pd@198Au : Au. These nanoparticles were synthesized using chemical reduction technique, one-pot method. In 103Pd-Au nanoparticles, the 103Pd : Pd radioactive core served the purpose of low-energy photon source, and the outer gold (Au) shell provided biocompatibility and protection and enhanced the radiation dose delivered by the process of radiosensitization. Additionally, 103Pd-Au nanoparticles were labeled with 198Au (high-energy beta emitter). In order to minimize the absorption of Auger and delta electrons by gold, the core size was kept at the range of 5–30 nm. The nanoparticles were synthesized with radiochemical yield of 87%. These nanoparticles were further functionalized with PEG; 103Pd : Pd@Au-PEG and 103Pd : Pd@198Au : Au-PEG nanoparticles were synthesized. In order to assess the therapeutic efficacy of both types of nanoparticles, a single dose of 1.6–1.7 mCi (2–4 μL) was intratumorally injected to the mice with prostate cancer tumors (PC-3 cell lines). Four weeks post radioactive injection, a delay in tumor growth by 56% and 75% was reported for 103Pd : Pd@Au-PEG NPs and 103Pd : Pd@198Au : Au-PEG NPs, respectively, with respect to the controls. Through biodistribution evaluation, the authors demonstrated that most of the nanoparticles were retained within the tumor, as more than 75% of the total radioactivity measured in the mice at the time of euthanasia was found there. Additionally, up to 16% of nanoparticles were found in the liver, 3% in the spleen, and less than 1% in other organs.Laprise-Pelletier et al.  synthesi125I. Initially, Ag+ ion was attached to the N atom of the bipyridine group present on 2,2′-bipyridine-based COF, and COF-Ag bioconjugate was formed. This bioconjugate was functionalized with PEG and radiolabeled with 125I, consequently resulting in the formation of PEG-COF-Ag-125I nanoparticles with radiolabeling yield of 94% and stability of more than 90% (after 7 days) in PBS and serum. The authors also evaluated the in vitro radiotoxicity of PEG-COF-Ag-125I nanoparticles on PC-3 cell lines with variable activity (0–200 Ci/mL). The decrease in the survival of PC-3 cells by 25.8% was reported. Furthermore, the therapeutic efficacy of the 125I radiolabeled nanoparticles was also evaluated. To evaluate the therapeutic efficacy, the mice were divided into three groups: i) control, injected with 50 μL of PBS; ii) 125I group, injected with 1 mCi of 125I in 50 μL of PBS; and iii) 125I-COF group, treated with 1 mCi of PEG-COF-Ag-125I radiobioconjugate. The radiopharmaceutical and PBS were injected intratumorally to the mice. The activity retention time was studied through SPECT/CT at 0.5, 10, 24, and 36 h p.i. The authors demonstrated that at 0.5 h p.i., signal intensity was 3.2 times higher at tumor site for 125I-COF group in comparison with 125I group. On average, 61.67% of PEG-COF-Ag-125I nanoparticles were retained in the tumor volume. Based on the data of time of retention of nanoparticles in the tumor volume, all three groups were reinjected with PBS, 1 mCi of 125I, and 1 mCi of PEG-COF-Ag-125I after 4 days; and the mice were euthanized 9 days after the first day of radioactive injection. For the 125I-COF group, the reduction in the tumor volume by about 63% in comparison with the initial size was reported. Additionally, an increase in the tumor size by factor of 2 for control and 125I group with respect to the initial tumor size was reported. The authors did not perform the biodistribution evaluation of the radio-NPs.Zhang et al.  used COF103Pd]AuPd radio-NPs using chelator-free radiolabeling technique. The [103Pd]Pd2+ was reduced in the presence of Au3+ and citric acid to form [103Pd]AuPd radio-NPs of 15-nm size and 23.5-nm hydrodynamic diameter. The radio-NPs were coated with a biocompatible polymer, poly (N-isopropylacrylamide) (PNIPAAm), resulting in the formation of hydrophobic [103Pd]AuPd radio-NPs of 37.3-nm diameter. The PNIPAAm-coated radio-NPs were further mixed with sucrose acetate isobutyrate (SAIB) or lactose octaisobutyrate (LOIB) in the presence of ethanol. A biocompatible, low-viscosity, injectable LOIB : EtOH radioactive “nanogel” containing [103Pd]AuPd was synthesized. The therapeutic efficacy of the radioactive nanogel was assessed on mice with syngeneic CT26 colorectal cancers. The mice were divided into three groups: i) control group: the intratumoral injection was mimicked by inserting a syringe needle into the tumor, and nothing was injected. ii) “Cold nanogel” group: 50 μL of LOIB : OH bioconjugate was injected into the tumor through intratumoral injection. iii) Treated group: 0.675 μCi (25 MBq) was injected into 50 μL of radioactive LOIB : EtOH-[103Pd]AuPd nanogel. The delay in the tumor growth after day 10 p.i. was reported in comparison with the control and cold nanogel groups. Further, the ex vivo biodistribution studies elucidated that up to 95%ID/g of injected radioactive nanogel was retained in the tumor post day 20 of injection and less than 0.01%ID/g of nanogel was found in the kidney, liver, spleen, and muscles of the mice. Additionally, the authors found no evidence of release of radioactivity from the LOIB : EtOH gel.Fach et al.  synthesi198Au}) nanoparticles using poly(amidoamine) (PAMAM) dendrimers and chloroauric (HAuCl4) acid for nanobrachytherapy applications. The steps involved in the synthesis of gold-dendrimer nanoparticles were formation and decomposition of dendrimer-amine[AuCl4] complex, followed by reduction of Au3+ to Au. Consequently, positively charged poly{197Au} nanoparticles of 10- to 50-nm size range were fabricated. The positive charge of these nanocarriers was expected to enhance the internalization of nanoparticles within the tumor cells. Furthermore, the 10- to 50-nm size range was used to take advantage of enhanced permeability and retention (EPR) effect. EPR effect is increased in accumulation of nanoparticles within the tumor due to the porosity and irregularity in the tumor microvasculature. The aqueous solution of poly{197Au} nanoparticles was irradiated with neutron beam, and poly{198Au} was obtained. The therapeutic efficacy studies of poly{198Au} were performed on C57BL/6J mice having B16F10 melanoma tumor model. At the time of treatment, mice were approximately 8 weeks old and had tumor size of 440 to 530 mm3. For therapeutic evaluation, the mice were divided into three groups, with each group having seven mice: i) Group A was administered 35 μCi of poly{198Au}, in PBS, intratumorally; ii) Group B received 74 μCi of poly{198Au} in PBS through intratumoral injection; and (iii) Group C was injected with 75 μL of PBS per mouse. The tumor size was monitored for 8 days post radioactive injection. Group A mice (treated with 35 μCi of poly{198Au}) showed a delay in tumor growth in comparison with the control (Group C). However, the difference was not statistically significant. Reduction in tumor growth by more than 45% was observed for Group B mice (injected with 74 μCi of poly{198Au}) in comparison with the control and Group A. The authors did not perform biodistribution studies.Khan et al.  synthesi198AuNPs), with a diameter of 4–10 nm, were synthesized by adding tris hydroxymethyl phosphine-aniline (P(CH2NHCH(CH3)-COOH)3 (a reducing agent) and GA to H198AuCl4  were injected intratumorally to the severely compromised immunodeficient (SCID) mice bearing prostate tumor (PC3 cells) xenografts. Each mouse was given an intratumoral injection of 408 μCi of GA-198AuNPs (30 μL). The tumor volume was monitored over a period of 30 days, and retardation in tumor growth for the treated group in comparison with the untreated group was reported. After 3 weeks of radioactive injection, the tumor volumes of treated groups were found to be 82% smaller than in the control group. Furthermore, even after 30 days of injection, on ex vivo analysis, radio-NPs were found in the tumor (20%ID), the liver (1%ID), and the carcass (18.5%ID)  . In rece198Au-EGCg. EGCg is a phytochemical extracted from green tea leaves and can be used to actively target laminin receptors (Lam 67R), which are overexpressed by the prostate cancer cells. In this study, i) the synthesis and characterization of 198Au-EGCg nanoparticles were reported; ii) the affinity of EGCg for laminin receptors and internalization of 198Au-EGCg through endocytosis was demonstrated; (iii) in vivo therapeutic assessment of 198Au-EGCg nanoparticles was performed. For in vivo therapeutic assessment, 136 μCi (30 μL) of 198Au-EGCg nanoparticles, with a diameter of 40–55 nm, were injected intratumorally to the mice bearing prostate tumor. The pharmacokinetic study results demonstrated that after 24 h of injection, approximately 72% of 198Au-EGCg nanoparticles were retained in the tumor. After 28 days of injection, the tumor size of the treated group was found to be 80% smaller than of the control group. The results of end-of-study biodistribution, conducted on day 42 post radioactive injection, showed that radio-NPs were retained in the tumor (34.7%ID), liver (2.5%ID), and carcass (18%ID).Shukla et al.  synthesi198AuNPs) was also assessed in the canine model (198AuNPs (diameter 12–15 nm) intratumorally. In order to obtain homogeneous distribution of a radiotherapeutic agent within the tumor volume, two to eight needles were inserted, and several injections of 100–200 μL were administered. Activity to be administered was selected as a function of tumor volume. The dogs were injected with activity in the range of 3 to 13.8 mCi of 198Au. This activity range corresponded to the biological effective dose of 50 (n = 2) and 150 Gy (n = 7). After 30 min of radioactive injection, scintigraphy scans were performed. In six dogs, the migration of nanoparticles to the bladder, urethra, and prostatic extra region from the prostate was observed. After 30 min of injection, only 53% of injected radio-NPs were retained in the prostate. Four weeks posttreatment, CT scan was performed to measure the tumor volume. The authors expressed the effectiveness of the treatment in terms of decrease in the tumor volume. A decrease in the tumor volume by 30%–50% was observed in two specimens, an increase in tumor size by 12%–26% was observed in two dogs, and for the remaining specimens, there was an increase or decrease of 3% in the tumor volume. The nanoparticles did not induce any sign of toxicity. The authors concluded that the therapeutic effectiveness of GA-198AuNPs in the canine model was compromised due to the limited retention of radio-NPs within the tumor volume. Hence, the influence of tumor vasculature and the lymphatic drainage on retention or leakage of nanoparticles need to be investigated before conducting clinical trials.The therapeutic effectiveness of GA-coated AuNPs  tumor cells. Hence, selective accumulation of MGF-198AuNPs within the tumor volume was achieved. The authors reported the following: i) the fabrication and characterization of MGF-198AuNPs; (ii) studies on stability of MGF-198AuNPs in vitro and in vivo and biodistribution studies; and (iii) studies on the evaluation of therapeutic efficacy of MGF-198AuNPs on mice bearing prostate tumors. In order to evaluate the in vivo stability, normal mice (N = 25) were given intravenous injection of 8.0 μCi/100 μL of MGF-198AuNPs and were euthanized at 30 min, 1 h, 2 h, 4 h, and 24 h post radioactive injection. All the important organs  were collected, and radioactivity accumulation in these organs was estimated. MGF-198AuNPs predominantly accumulated in the spleen and liver clearance through hepatobiliary pathway, and almost no uptake occurred in the blood and lungs. In order to evaluate the selective accumulation of MGF-198AuNPs, due to the glucose functionality of MGF, the authors performed a study on the retention of radiopharmaceuticals within the tumor. Mice bearing PC-3 tumor (N = 5) were administered with a single dose of 4 μCi/30 μL of MGF-198AuNPs for each tumor through intratumoral injection. The mice were euthanized at an interval of 30 min, 1 h, 2 h, 4 h, and 24 h post radioactive injection; and tumors and the organs of interest  were excised. Radioactivity accumulation in tumor and different organs was estimated in terms of %ID/organ. At 30 min and 24 h p.i., 80.98% ± 13.39% and 79.82% ± 10.55% of MGF-198AuNPs were respectively found to be accumulated in the tumor, whereas liver increase from 4.05% ± 5.27% (at 30 min) to 10.65% ± 8.31% (at 24 h) was reported. Additionally, low uptake of radio-NPs was also found in feces (0% at 30 min and 2.2% ± 4.5% at 24 h) and the stomach (0.10% at 30 min and 0.02% at 24 h), and no noticeable uptake was found in the lungs, blood, and other organs. Lastly, the authors also performed a detailed study to evaluate the therapeutic efficacy of MGF-198AuNPs. Mice bearing PC-3 tumors were divided into three groups: i) Group A, tumor volume ranging from 0.15 to 0.2 cm3; ii) Group B, mice with tumor volume about 0.43 cm3 were injected with a single dose of 160 μCi/30 μL of MGF-198AuNPs per tumor through intratumoral injection; and iii) Group C, mice with 0.15 to 0.2 cm3 of tumor size were injected with 30 μL of saline intratumorally and served as control. The tumor volume was monitored for 3 weeks. Post 7 days of injection, a decrease in the tumor volume for Groups A and B was observed. After 2 weeks of injection, a decrease in the tumor volume by twofold with respect to control was reported for the treated groups. Three weeks post radioactive injection, there was an increase in the tumor volume by fivefold for Group C; Group A = 0.18 ± 0.17 cm3 and Group B = 0.22 ± 0.02 were reported. Furthermore, after 3 weeks, 69.70 ± 14.40%ID was found to be retained in the tumor, 6.80 ± 5.9%ID in the carcass, and 1.44 ± 2.97%ID in the liver.In the most recent publication from this group , they us4 and GA resulted in the formation of AuNPs@GA. AuNPs@GA nanocarriers were made radioactive through neutron activation, and 198AuNPs@GA were obtained. Radiotherapeutic efficacy, biodistribution, and toxicity studies were performed on mice bearing H460 tumor. Suspension of 103 μCi (injection volume = 100 μL) of 198AuNPs@GA nanoparticles per mouse was administered intratumorally to the mice bearing H460 tumors, and the tumor volume was monitored for 2 weeks. Toxicity caused by administration of 198AuNPs@GA nanocarriers was evaluated in terms of loss in body weight. Less than 20% decrease in body weight was found post 4 days of radioactive injection; and post 7 days of injection, the body weight was recovered. Hence, the authors effectively showed that 198AuNPs@GA are safe for treatment. In order to perform biodistribution studies, mice were euthanized, and important organs  were collected. The authors also collected urine and feces. Post 7 days of injection, a decrease in the tumor volume by more than 90% was observed in the 198AuNPs@GA-treated group in comparison with the controls and mice injected with non-radioactive nanoparticles. Even after 2 weeks of radioactive injection, 50% of the nanoparticles were found to be accumulated in the tumor and 8.9% in the liver. Furthermore, clearance of 198AuNPs@GA was observed in urine and feces.Lin et al.  fabricat177Lu using a macrocyclic complex: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and NPs)were functionalized with PEG and panitumumab (an antibody) to target the epidermal growth factor receptors (EGFRs). The EGFRs are overexpressed by the BC tumor cells. Radio-NPs were divided into two categories: i) targeted—functionalized with PEG and panitumumab-177Lu-T-AuNP; and ii) non-targeted—functionalized with PEG but not panitumumab-177Lu-NT-AuNP. A single dose of 4.5 MBq of both targeted and non-targeted nanoparticles in 30 μL of saline was administered through intratumoral injection into the mice carrying subcutaneous human BC cells. Both targeted and non-targeted 177Lu radiolabeled AuNPs were found to be capable of delaying tumor growth for more than 90 days, and no organ toxicity caused by these nanoparticles was reported. In the treated groups, inhibition of tumor growth by a factor of ≈30 in comparison with the untreated groups was reported. The amount of nanoparticles that was retained within the tumor was evaluated by performing SPECT/CT imaging at 1 and 48 h post radioactive injection. Ex vivo analysis was also done to assess the distribution of 177Lu-T-AuNP and 177Lu-NT-AuNP in different organs. Post 1 h of injection, most of the radio-NPs were confined within the tumors, and migration of this radioactive out of tumors was observed at 48 h. Furthermore, the authors reported that high concentrations of both targeted and non-targeted nanoparticles, >300%–400%ID/g, accumulated within the tumors after 1 h of intratumoral administration. Hence, no significant impact of active targeting of 177Lu-AuNP was observed in retaining the AuNPs within the tumors. Less than 3%ID/g radioactivity migrated to the liver and spleen, and its value increased by two- to fivefold post 48 h of injection, whereas the radioactivity found in other organs was less than 0.5%ID/g.Yook et al.  evaluate177Lu-DOTA to synthesize 177Lu-AuNPs. These nanoparticles were further functionalized with trastuzumab antibodies using PEG. Initially, the PEG chains were linked on the AuNPs, and the trastuzumab molecules were attached on these chains. The nanoparticles were categorized into two groups: i) targeted—nanoparticles functionalized with trastuzumab (trastuzumab-AuNP-177Lu); and ii) non-targeted—nanoparticles not functionalized with trastuzumab (AuNP-177Lu). In order to assess therapeutic effectiveness of these nanoparticles, 3 MBq (≈81 μCi) was administered intratumorally in mice bearing BC tumors. The tumor growth was monitored for 16 days. The targeted nanoparticles (trastuzumab-AuNP-177Lu) were reported to be 1.8 times more efficient in inhibiting tumor growth in comparison with the non-targeted nanoparticles (AuNP-177Lu) and 2.2 times in comparison with the untreated group. No significant tissue toxicity was reported by the authors for both targeted and non-targeted treatments. Additionally, the authors provided no information on the amount of nanoparticles that migrated to the liver and spleen.Cai et al.  radiolab199Au radio-NPs with an average particle size 11 nm and hydrodynamic size of about 30.2 nm. Cyclic  [f(RGDfK)] peptide was used as both a stabilizing agent and a reducing agent for the synthesis of 199Au-c(RGDfK) nanoparticles to target integrin αvβ3 receptors for nanobrachytherapy applications. Additionally, non-targeted 199Au nanoparticles were also synthesized by labeling 199Au nanoparticles with scrambled sequence of RGD cyclic ]. The non-targeted 199Au-c(RGKfD) nanoparticles were used as control. The authors characterized the nanoparticles using numerous analytical techniques to evaluate the particle identity, size, in vitro stability, compatibility to biological medium, and suitability for clinical use. The biodistribution studies were conducted in C57BL/6 mice bearing melanoma tumors after intratumoral administration of 199Au-c(RGDfK) nanoparticles. The non-targeted 199Au-c(RGKfD) nanoparticles were also injected intratumorally to another group of C57L/6 mice having melanoma tumors and were used as control. The mice were euthanized at 24, 72, and 192 h post radioactive injection, and samples of normal tissues and tumor were collected. At 24 h p.i., a high percentage of administered radioactive 199Au nanoparticles (both targeted and non-targeted) were retained within the tumor volume. The uptake of targeted 199Au-c(RGDfK) nanoparticles (497 ± 56%ID/g) was reported to be higher than that of non-targeted (400 ± 67%ID/g). Between 24 and 192 h post intratumoral injection, a gradual decrease in radioactivity, accumulated in the tumor, was observed for both targeted and non-targeted 199Au nanoparticles. Additionally, at 192 h p.i., twofold higher retention of the targeted 199Au-c(RGDfK) nanoparticles (375 ± 78%ID/g) in comparison with non-targeted 199Au-c(RGDfK) nanoparticles (182 ± 23%ID/g) was observed. Consequently, higher radioactivity was found in the blood for non-targeted nanoparticles in comparison with the targeted nanoparticles, indicating their leakage from the tumor. Post 120 h of injection, the uptake of targeted 199Au-c(RGDfK) (≈4%ID/g) in the liver and kidney was found to be three times lower than of non-targeted 199Au-c(RGKfD) (≈12%ID/g) nanoparticles. The uptake in the spleen (≈2%ID/g) was nearly equal for both targeted and non-targeted 199Au nanoparticles. The uptake of radio-NPs in the remaining organs was less than 1%ID/g. The therapeutic efficacy of these targeted and non-targeted 199Au nanoparticles was evaluated on melanoma-bearing C57BL/6 mice. The mice with tumor size approximately 150 mm3 were divided into five sets (five mice per set). Each group was given a single intratumoral injection of saline, non-radioactive Au-c(RGDfK), 2 MBq of 199Au-c(RGDfK), 5 MBq of 199Au-c(RGDfK) nanoparticles, or 10 MBq of 199Au-c(RGDfK) nanoparticles. The first two groups were used as control. Furthermore, the tumor volume and body weight of the mice were monitored for 15 days. A significant delay in tumor growth was observed in mice injected with 2, 5, or 10 MBq of 199Au-c(RGDfK) nanoparticles in comparison with the control.Chakravarty et al.  synthesi131I. The MNPs were radiolabeled with Ag-I two-step method. First, Ag+ ions were chelated by MNPs, and 131I ions were attached to Ag+ ions to form MNP-Ag-131I nanoparticles  with 99% radiolabeling yield. The authors further evaluated the solubility and/or stability of MNP-Ag-131I in demineralized water (DI water), PBS, and serum. Additionally, the in vitro biocompatibility was tested in PC-3 prostate cancer cells, and no cytotoxicity was observed. In order to evaluate the in vivo therapeutic efficacy of MNP-Ag-131I nanoparticles, the mice were divided into three groups: i) control, ii) 131I group, and iii) MNP-Ag-131I-treated group. On day 1, the 131I group and MNP-Ag-131I-treated group were injected with 1 mCi of 131I and MNP-Ag-131I through intratumoral injection; and radiopharmaceutical retention within the tumor was observed through SPECT and Cherenkov radiation. On day 3, through intratumoral injections, control, 131I group, and MNP-Ag-131I-treated group were injected with 20 mL of PBS, 500 mCi of 131I in 20 mL of DI, and 500 mCi of MNP-Ag-131I in 20 mL of PBS. The mice were euthanized after 7 days of radioactive injection, and tumor and other important organs were collected. The MNP-Ag-131I-treated group had a tumor volume equal to the initial volume, whereas the control and 131I-treated group had tumor size 1.5 times larger in comparison with the initial volume.Sheng et al.  synthesi131I and activated with anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) antibodies and bovine serum albumin (BSA) for the treatment of anaplastic thyroid cancers (APCs). The radiolabeling of the MSNPs was performed using Chloramine-T method, resulting in the formation of 131I-BSA-MSNPs-anti-VEGFR2 radioactive nanocarriers. In vitro cellular uptake of 131I-BSA-MSNPs-anti-VEGFR2 in human thyroid carcinoma FRO cell lines was evaluated through confocal imaging, and time-dependent cellular uptake was evaluated by measuring radioactivity using gamma counter. The therapeutic efficacy of radioactive 131I-BSA-MSNPs-anti-VEGFR2 was tested on mice bearing FRO tumor cells. The radiopharmaceutical retention within the tumor was measured through SPECT/CT imaging. The mice were divided into four groups: control, injected with PBS; and 131I-BSA-MSNPs, Na131I, and 131I-BSA-MSNPs-anti-VEGFR2-treated groups (n = 3). Each group was administered with radioactivity of 74 MBq (50 μL) through intratumoral injection. A gradual increase in the tumor volume was reported for all the groups except 131I-BSA-MSNPs-anti-VEGFR2-treated group.Zhang et al.  synthesi131I and activated with twin arginine translocation (TAT) peptide. In order to construct 131I-AuNPs-TAT radiopharmaceuticals, first, AuNPs (diameter = ≈8.36 nm) were prepared. Later, AuNPs were functionalized with amino-poly(ethylene glycol)-thiol (HS-PEG2000-NH2) to prepare AuNPs-PEG, and they were conjugated with TAT peptide to prepare AuNPs-TAT. Lastly, through iodogen-catalyzed procedure, AuNPs-TAT was radiolabeled with 131I to synthesize 131I-AuNPsTAT radiopharmaceutical with radiolabeling yield of 96.5% and radiochemical purity above 78%. In vitro experiments on radiocytotoxicity, estimating the rate of apoptosis and suppression of tumor cell proliferation, were performed using cell counting kit-8 (CCK-8) assay by exposing human colon cancer (HCT-116) cells to 131I-AuNPs-TAT radiopharmaceutical. The authors concluded that, after the addition of TAT peptide and AuNPs, 131I-AuNPs-TAT was internalized by the cell nuclei and caused short-term and long-term damage to the tumor cells. From the results of in vitro studies, the authors concluded that 500 μCi/mL of 131I-AuNPs-TAT (composed of AuNPs = 100 μg/mL and TAT = 10 g/mL) is appropriate for therapeutic studies. Mice bearing HCT166 tumors were used to evaluate the therapeutic efficacy of 131I-AuNPs-TAT. Prior to treatment, SPECT/CT imaging was used to monitor the metabolic distribution of intratumorally administered 131I-AuNPs-TAT. The authors reported that about 20.09% of 131I-AuNPs-TAT was retained at the site of injection after 36 h. Post SPECT/CT imaging, mice were administered with 500 μCi/mL (per mouse) of 131I-AuNPs-TAT through intratumoral injection. After 18 days of radioactive injection, reduction in tumor size by 79.95% was reported for the 131I-AuNPs-TAT-treated group, whereas in the untreated group, the tumor grew to 8.08 times the original tumor size. The authors concluded that the presence of TAT and AuNPs i) internalized the radiopharmaceutical to the nuclei of the tumor cells, which elevated the DNA damage; and ii) the high-energy beta particles emitted from 131I on interaction with Au produced low-energy X-rays—this further reduced the cold spots and induced a strong immune response.Su et al.  synthesi211At followed by 225Ac, mostly radiolabeling AuNPs with small diameter (from 5 to 15 nm). The most interesting finding among the collected studies was the fact that intratumoral injection likely leads to a better outcome in terms of cancer ablation compared with intravenous injection. When it comes to Auger emitters, palladium comes into the scene more often, either cold palladium combined with gold in the core of nanoparticles or 103Pd as a radionuclide. Other Auger emitters that are also used are 111ln and 125I, and the intratumoral injection was the chosen route for administering the nanoparticles in all studies recruited for this paper. Interestingly, the tumor ablation appears to be higher than alpha emitters, with tumors decreasing in size from 56% to 75% among the recruited studies. Finally, beta emitters are likely the most effective in ablating solid tumors, with tumor size decreasing more than 80% in various preclinical studies. The most commonly used radionuclides are 198Au and 199Au followed by 131I and 177Lu. Again, the administration route for the nanoparticles was the intratumoral injection.From the data collected regarding alpha emitters in preclinical studies, it is possible to observe that the most commonly used radioisotope is Among the studies recruited for this paper, most of the authors synthesize the nanomaterials, making use of some sort of targeting strategy in order to enhance the tumor localization of the nanoparticles, apart from the intratumoral injection, which also contributes in this regard. However, targeting strategies are more utilized when the authors use beta emitters. Biodistribution studies were performed more often with beta emitters than alpha- or Auger-emitting radionuclides. In this matter, it is of utmost importance to carefully follow the pharmacokinetics of radioactive nanomaterials in order to avoid side effects and non-specific radioactive damage to healthy cells; therefore, researchers should work with novel strategies related to theranostic radioactive nanomaterials. Targeting strategies, i.e., mAb and tissue-specific receptor ligands, are very useful for concentrating therapeutic agents inside the tumor tissues; thus, they should be taken into consideration by those working with this kind of biomaterials. via activation of the innate and adaptive immune system  loaded with immunoadjuvants to study the abscopal effect of local RT . Lewis l131I and injected intratumorally. This creates in situ gelation to confine 131I within the tumor and alleviates tumor hypoxia (131I was added with CpG oligonucleotide (immunostimulator) administered intratumorally and combined with systemic checkpoint blockade therapy (CTLA-4 antibodies), it leads to local tumor eradication as well as increased systemic immune response to inhibit distal metastasis and tumor recurrence.There was one attempt to administer both radiation and an immune stimulator directly to the tumor. Sodium alginate formulation containing catalase was labeled with  hypoxia . They alRadiation in the form of nanoparticles is being investigated for intratumoral administration to reduce the side effects to normal tissues, and similarly, immunotherapeutic drugs are being administered intratumorally to avoid immune-related adverse events. The therapeutic combination of nanobrachytherapy and intratumoral immunotherapy has great potential to achieve higher therapeutic index in a synergistic manner. They may deliver larger doses of therapeutics to the tumor, reduce normal tissue toxicities of systemic delivery, eradicate distal malignant cells owing to the enhanced abscopal effect, and enhance the efficacy of immunotherapy as well as RT for multiple disease sites and larger patient base.Radio-NPs are emerging as promising radiotherapeutic agents for cancer treatment and are being probed as a replacement to seed-based BT. Prior to using radio-NPs for RT applications, accurate dosimetric simulations are needed in order to determine the dose distribution within the tumor volume and the surrounding normal tissues. Monte Carlo simulation techniques can be used efficiently to determine the energy or dose distribution within the region of interest. As per our knowledge, only three studies addressing the problem of dosimetry for nanobrachytherapy using radio-NPs have been published so far. The main highlights of these three studies are briefly discussed below.103Pd : Pd@Au were synthesized  was compared with the dose distribution obtained for the conventional millimeter-sized, low-dose BT: 103Pd seed. A sharper dose fall in the isodose curves estimated for a single injection of radio-NPs was reported in comparison with the conventional BT seed. This sharper dose fall was attributed to the attenuation of photons by the gold atoms present in the “cloud” of NPs. The authors stressed that this feature can be useful in effectively sparing organs at risk and delivering high doses of radiation to the tumor tissues, as NPs deposit very high doses of radiation in their immediate vicinity. The TEM images of the xenograft tumor cells were used to simulate the energy deposited by 103Pd : Pd@AuNPs at the microscopic level. The Monte Carlo simulation was conducted in three steps: i) the TEM images representative of microdistribution of NPs were selected and digitized. These digitized images were virtually placed in the middle of 50-μm3 cubical phantom. ii) Nanoconstructs (r = 25 nm) with Pd core (r = 5 nm) coated with a thick layer of gold (r = 20 nm) were simulated. These nanoconstructs were positioned in the resampled TEM images, placed in the cubical phantom. iii) Emitted photons and electrons produced in the interactions were simulated, and energy deposition maps were computed. From the computed dose distribution, it was found that the highest dose deposition occurred in the immediate vicinity of the NPs. That is, the electrons escaping from the NPs lost most of their energy in a very short range, and almost no NPs were found in the nuclei. The same microdosimetric approach was used to quantify the radiosensitization effect induced by gold. In this case, two simulations were performed, considering i) 103Pd : Pd core coated with gold and ii) 103Pd : Pd not coated with gold. The estimated energy deposition map was reported as a ratio of energy-deposited values (energy deposited by radio-NPs coated with gold/energy deposited by radio-NPs not coated with gold). The ratio of energy-deposited value quantified the dose enhancement effect due to the presence of gold. Enhancement in dose by factor of 25, in the immediate vicinity of the NPs coated with gold, was observed, whereas for regions 2 μm further from the radio-NPs, no radiosensitization effect was observed. Hence, the radiosensitization effect was found to be extremely localized around the gold-coated radio-NPs. Based on the microdosimetric results, the authors concluded that reactive oxygen species (ROS) can be the main factor responsible for cell killing and observed strong tumor control effect  homogeneously distributed within the tumor. This simple model consisted of spheres representing the following: tumor (radius (r) = 0.4 cm) located within the prostate (r = 2 cm), prostate, bladder (r = 3.5 cm), and rectum (r = 1.5 cm). Dose distribution was estimated for tumor and other organs at risk , assuming that 10 mCi of 198AuNPs/199AuNPs was homogeneously distributed within the tumor volume. The authors reported that for both 198AuNPs and 199AuNPs, the maximum dose was deposited at the center of the tumor and decreased rapidly towards the tumor prostate interface and other surrounding organs. Owing to the high-energy beta emissions from 198Au, high dose rates were reported for 198Au at a) center of the tumor, 12 Gy/h; b) prostate tumor periphery, 1.46 Gy/h; c) prostate periphery, 0.1 Gy/h; d) center of the bladder, 0.013 Gy; and e) center of the rectum, 0.026 Gy/h. On the other hand, due to low-energy beta emissions for 199Au, for the same locations, the dose rates were 1.6, 0.53, 0.26, 0.0013, and 0.004 Gy/h. Based on these findings, the authors concluded that 198AuNPs are suitable for the treatment of solid tumors and that 199Au can be used for imaging purposes.Al-Yasiri et al.  used MCNin silico dosimetry study (103Pd (Auger emitter), 153Sm (medium energy beta emitter), and 198Au (high-energy beta emitter). Initially, the dosimetric characteristics of 103Pd, 153Sm, and 198Au were evaluated using single cell dosimetry  without depositing enough energy. Second, the estimated cell survival curves were found to be in good agreement with the experimental results published in literature. Lastly, we evaluated the impact of i) tumor size, ii) tumor type, and iii) amount of injected activity on the cell survival curves. We found that 153Sm and 198Au effectively ablated tumor cells for all three cases with minimum injected activity (≤20 MBq), whereas for 103Pd, higher radioactivity was required to achieve a similar effect. Hence, we concluded that for radioresistant, large size (≈1 cm3) and rapidly growing tumors, 153Sm and 198Au can be conclusively used as nanobrachytherapeutic agents, whereas 103Pd is only suitable for small-size (≈0.3 cm3) tumors that have injected activity ≥60 MBq.In one of our recent ry study , we replry study , 15, 100ry study  and EGSnry study  MC code.osimetry . It was The application of interstitial BT is impeded by several posttreatment adverse effects or symptoms and the associated operational and logistical complications. The emerging nano-platforms can be used to efficiently deliver radiopharmaceuticals to the tumor. In comparison with free radioisotopes or radioisotopes functionalized with single tumor-specific biomolecules, nanoparticles can be loaded with higher doses of radioactivity, and multiple radioisotopes can be accommodated within a single nanoparticle. Moreover, these nanocarriers can also provide several additional functions, for instance: i) photothermal effect, ii) load chemotherapeutic drug, iii) radiosensitization in case of high-Z nanoparticles, and iv) real-time tumor imaging. Hence, they can be helpful in improving the efficacy or optimizing the therapeutic planning of internal RT or systemic therapy. Radio-NPs, injected intratumorally, can directly deliver radiation dose to the tumor like BT; and this technique is termed as nanobrachytherapy.In this article, we review the recent progress in the radiosynthesis of the nanoparticles and their use for nanobrachytherapy applications. Recent progress on the i) radiosynthesis methods, ii) selection of a radionuclide for nanobrachytherapy application, iii) modes of internalization of nanocarriers, and iv) the most recent preclinical and dosimetric studies on BT are discussed.The intratumoral (i.t.) injection of radio-NPs for nanobrachytherapy applications is associated with several challenges and shortcomings. The two main obstacles that have impeded the clinical translation of radio-NPs are i) leakage of fraction of injected radio-NPs from the tumor and ii) inhomogeneous distribution of radioactivity within the tumor post i.t. injection.Intratumoral retention of radio-NPs is crucial for the therapeutic effectiveness of nanobrachytherapy application and must be maximized. It also reduces the risk of irradiating normal tissues or healthy organs . Both inhomogeneous intratumoral radioactivity distribution and leakage of radio-NPs from the tumor post injection are caused by irregular tumor vasculature, variable blood and lymph flow, and pressure gradients. Since tumors are unique, the radio-NP leakage ratio and distribution of NPs within the tumor may vary from patient to patient. This will probably make treatment planning and dosimetric computations complicated and challenging. The tumor retention of radio-NPs can be improved by i) functionalizing surface of radio-NPs with tumor-specific biomolecules or ii) co-injecting biocompatible polymers that sequester NPs within the tumor, along with radio-NPs. The delivery systems, injected intratumorally, that can homogeneously distribute radioactivity throughout the tumor volume with minimal leakage have not been developed yet.Consequently, for clinical translation of intratumorally injected radio-NPs for nanobrachytherapy applications, the injected radio-NPs should i) have high intratumoral retention and ii) homogeneously distribute radioactivity throughout the tumor volume.in vivo microscopic distribution of radio-NPs, ii) complex cell geometry, and iii) distribution of radio-NPs near and within the nucleus. The computational model should also include all possible physics processes that are susceptible to participate in radiation dose distribution at the microscopic scale.Hence, more comprehensive biodistribution studies are required to understand and control the excretion routes of radio-NPs. Furthermore, intratumoral distribution and diffusion of NPs depend on i) tumor architecture and its density; ii) interstitial fluid pressure; iii) tumor vasculature, blood flow, and lymph flow; and iv) specifics of extracellular matrix of the tumor. These factors must be investigated in a wide range of tumors in order to reduce the inhomogeneity in the intratumoral radioactivity distribution. Lastly, for accurate computation of dose distribution at the cellular and subcellular levels within a tumor injected with radio-NPs, the computational model should consider i) BS: initial idea, conceptualization, data collection, and manuscript preparation. LF: manuscript preparation. VT: manuscript preparation. SS: initial idea, basic framework, review of the manuscript, and supervision. TF: supervision and manuscript review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.